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WO1993015097A1 - Reactif et procede pour la determination de l'activite d'enzymes glycohydrolitiques - Google Patents

Reactif et procede pour la determination de l'activite d'enzymes glycohydrolitiques Download PDF

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Publication number
WO1993015097A1
WO1993015097A1 PCT/US1993/000988 US9300988W WO9315097A1 WO 1993015097 A1 WO1993015097 A1 WO 1993015097A1 US 9300988 W US9300988 W US 9300988W WO 9315097 A1 WO9315097 A1 WO 9315097A1
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Prior art keywords
glycohydrolytic
pyrene
enzyme
carbohydrate
compound
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PCT/US1993/000988
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English (en)
Inventor
Mark R. Pope
Christopher Bieniarz
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Abbott Laboratories
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Priority to KR1019940703768A priority Critical patent/KR950701255A/ko
Publication of WO1993015097A1 publication Critical patent/WO1993015097A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
    • C07H15/20—Carbocyclic rings
    • C07H15/24—Condensed ring systems having three or more rings
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2334/00—O-linked chromogens for determinations of hydrolase enzymes, e.g. glycosidases, phosphatases, esterases
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/924—Hydrolases (3) acting on glycosyl compounds (3.2)
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S436/00—Chemistry: analytical and immunological testing
    • Y10S436/80—Fluorescent dyes, e.g. rhodamine
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S436/00—Chemistry: analytical and immunological testing
    • Y10S436/807—Apparatus included in process claim, e.g. physical support structures
    • Y10S436/808—Automated or kit

Definitions

  • the present invention relates to the determination of glycohydrolytic enzymes.
  • the present invention relates to pyrene-trisulfonic acid derivatives of carbohydrates and the use thereof in assays for the determination of glycohydrolytic enzymes such as ⁇ -glucuronidase.
  • glycohydrolytic enzymes are a class of enzymes which have been employed in various diagnostic applications, such as for measuring PMN phagocytic activity and periodontal disease activity, for classifying bacteria, and as a component of conjugates in enzyme immunoassay procedures.
  • determination of glycohydrolytic enzymes involves the use of a hydrophobic substrate.
  • substrates include synthetic compounds such as phenolphthalein glucuronide and methylumbelliferyl glucuronide, and metabolic products such as the glucuronides of steroids and bilirubin.
  • hydrolytic enzymes such as ⁇ -glucuronidase, for highly polar substrates has not been described.
  • esters show violet fluorescence ( ⁇ ma ⁇ 390 and 408 nanometers) when excited below 385 nanometers, they do not fluoresce when excited at 460 nanometers.
  • hydrolysis of such esters results in the formation of an anion of the formula [II]:
  • [IV] The methyl(aryl-tri-0-acetyl-(H ⁇ )-D-glucopyranosid)-uronates described by Bollenback, et al., include those where R in the formula [V] is phenyl, 1-naphthyl and 2-naphthyl, and die alpha anomers of the compounds of the formula [IN] and [N] where R is o-chlorophenyl, p-chlorophenyl, o-bromophenyl, p-bromophenyl, o-iodophenyl, o- tolyl, m-tolyl, p-tolyl, o-hydroxyphenyl, 1-naphthyl, 2-naphthyl, methyl gentisate and o-nitrophenyl are also described.
  • the present invention provides novel pyrene-trisulfonate derivatives, the synthesis of such derivatives, and the use thereof for the determination of glycohydrolytic enzyme activity.
  • the pyrene- trisulfonate derivatives of the present invention are of the general formula [VI]:
  • R is a carbohydrate, or a carbohydrate derivative, which is capable of binding to a glycohydrolytic enzyme and comprises from 1 to 10 linked monosaccharide units, and wherein Mi, M 2 , and M 3 , which can be the same or different, are an alkali metal, hydrogen, ammonium, alkyl-substituted ammonium, or aryl-substituted ammonium.
  • R is ⁇ -D-glucuronide, ⁇ -D-giucoside, ⁇ -D- galactoside, ⁇ -D-maltoside, a monosaccharide, or a disaccharide.
  • the compound of formula [VI] does not fluoresce when excited with light having a wavelength of 460 nm, whereas the aglycon moiety thereof does fluoresce at such wavelength and, accordingly, is especially useful for the determination of glycohydrolytic enzymes.
  • glycohydrolytic enzymes include, but are not intended to be limited to, ⁇ - glucuronidase, ⁇ -glucosidase, ⁇ -galactosidase, and the like.
  • the method for determining a glycohydrolytic enzyme according to the present invention comprises the steps of:
  • a test solution comprising a test sample containing the glycohydrolytic enzyme and the compound of formula VI, wherein the glycohydrolytic enzyme binds to R and causes the hydrolysis of the compound to result in the formation of free 8- hydroxy-l ,3,6-pyrene trisulfonate as a function of, and which can be correlated to, the amount of the glycohydrolytic enzyme present in the test sample; and (b) measuring and correlating either the intensity of fluorescence, or the optical density, of the test solution to the presence or amount of the glycohydrolytic enzyme in the test sample.
  • the test sample can be selected from a variety of fluids or tissue, which include, but are not intended to be limited to, plasma, serum, ascites fluid, lymphatic fluid, cerebral spinal fluid, nipple fluid discharge, urine, gingival crevicular fluid, and the like.
  • the test sample can also be selected from a variety of environmental or microbiological samples such as sewage, water, soil, oil, bacterial cultures, and the like, for the detection of bacterial glycosidases (Acta
  • test sample can be diluted in a suitable diluent buffer,- such as phosphate buffered saline with serum components, to provide a sample volume that is required by a particular assay.
  • a suitable diluent buffer such as phosphate buffered saline with serum components
  • the test solution is excited with radiant energy having a wavelength of from between about 390 nm and about 490 nm wherein the selection of the appropriate wavelength will depend upon the pH of the test solution.
  • the test solution is excited with radiant energy having a wavelength of from between about 410 nm for test solutions having a pH of about 5.0 or less and about 454 nm for test solutions having a pH of about 9.0 or greater.
  • Such radiant energy causes 8-hydroxy-l,3,6-pyrene trisulfonate to fluoresce wherein the amount of fluorescence, which is measured at a wavelength of from between about 500 nm and about 530 nm, preferably at about 515 nm, can be correlated to the amount of the glycohydrolytic enzyme present in the test sample.
  • the optical density of the test solution can be measured and correlated to the amount of the glycohydrolytic enzyme present in the test sample.
  • such increase in optical density is measured at a wavelength of from between about 390 nm and about 490 nm wherein the selection of the appropriate wavelength will depend upon the pH of the test solution.
  • the optical density is measured at a wavelength of from between about 410 nm for test solutions having a pH of about 5.0 or less and about 454 nm for test solutions having a pH of about 9.0 or greater.
  • the measured changes in the optical density are the result of the liberated form of 8-hydroxy-l,3,6-pyrene sulfonate and can be correlated to the amount of the glycohydrolytic enzyme present in the test sample.
  • a preferred embodiment of the present invention provides for the synthesis of pyrene-(l,3,6-trisulfonic acid)-8- ⁇ -D- glucuronide of the formula [VII] :
  • the determination of ⁇ -D-glucoronidase is particularly useful for the diagnosis of periodontal disease wherein an elevated activity thereof in gingival crevicular fluid indicates acute inflammatory response.
  • the compounds according to the present invention are significantly more soluble in water and aqueous systems than those substrates previously described, particularly for ⁇ '-glucuronidase, and a shift in the absorption and emission spectrum between the protonated form of the dye and the glycosides enables kinetic analysis at a pH of less than about 7.0.
  • the present invention also provides other pyrene-(l ,3,6-trisulfonic acid) derivatives and the use thereof for determining other enzymes.
  • Figure 1 illustrates an ' end-point fluorometric analysis for ⁇ -glucuronidase ( ⁇ G) and the amount of 8-hydroxy-l,3,6-pyrene trisulfonate (Pyr) formed as an end product (BG is expressed as enzyme activity using phenophthalein ⁇ -D-glucuronic acid [uIU]).
  • Figure 2 illustrates a kinetic fluorometric analysis of ⁇ - glucuronidase hydrolysis of 1 ,3,6-pyrene trisulfonate-8- ⁇ -D- glucuronide.
  • Figure 3 illustrates a kinetic absorption analysis of ⁇ - glucuronidase hydrolysis of 1 ,3,6-pyrene trisulfonate-8- ⁇ -D- glucuronide.
  • the pyrano ring of the compounds that are described herein is represented as a six membered heterocyclic ring composed of five carbons and one oxygen, with a line extending above and below each of the carbons. Substituents other than hydrogen are shown at the end of each line that is above or below the relevant carbon to indicate that the isomer represented has that substituent above or below, respectively, the carbon to which it is bonded.
  • Hydrogens attached directly to the carbons of the pyrano ring are not shown but, of course, are present wherever required to satisfy the four valences of the carbons-
  • conventional numbering is used, the carbons of the pyrano ring being numbered in ascending order clockwise around the ring as viewed from above the paper on which it is represented, starting with the carbon adjacent the oxygen of the ring.
  • the compound of formula VII it is the 1 carbon of the * pyrano ring to which the pyrene sulfonate is bonded through an ether linkage, and the bond from the 1 carbon to the oxygen of the ether linkage is above the pyrano ring.
  • the 6 carbon is exocyclic, and is attached to the 5 carbon.
  • the compound 1,3,6- pyrene trisulfonate-8- ⁇ -D-glucuronide [VII] is produced as described in Example 1 hereinbelow, and can be used to conduct a kinetic determination of, for example, ⁇ -glucuronidase activity.
  • Such determination can be made by adding a 10 ⁇ L sample which contains ⁇ -glucuronidase to a 0.2 mL portion of a 0.3 mM solution of 1,3,6-pyrene trisulfonate-8- ⁇ -D-glucuronide [VII] in 0.1 M sodium acetate in deionized water, mixing thoroughly, and injecting the mixed sample into a fluorimetric flow cell which is temperature controlled at about 22.5°C while exciting at 410 nm and measuring emission at 515 nm. The initial rate of increase of fluorescence is proportional to the ⁇ -glucuronidase activity in the sample.
  • a standard curve can be prepared from data derived by carrying out the procedure, using solutions of known, and varying, ⁇ - glucuronidase activity, and the standard curve can be used to determine ⁇ -glucuronidase activity from fluorescence data concerning unknown samples.
  • the 1,3,6-pyrene trisulfonate-8- ⁇ -D-glucuronide [VII] can also be used to conduct a determination of ⁇ -glucuronidase activity by a procedure which involves determining a colorimetric endpoint. This determination can be made by adding a 50 ⁇ L sample which contains ⁇ -glucuronidase to 0.2 mL of a 0.3 mM solution of 1,3,6- pyrene trisulfonate-8- ⁇ -D -glucuronide [VII] in a 0.1 M solution of sodium acetate in deionized water, mixing thoroughly, incubating at 37°C for 15 minutes, quenching the reaction by adding 1.0 mL of a 0.25 M solution of glycine, pH 10.5, in deionized water, and measuring the optical density at 460 nm.
  • optical density is proportional to the ⁇ -glucuronidase activity of the sample.
  • a standard curve can be prepared from data collected from solutions containing known amounts of 8-hydroxy-l,3,6-pytene trisulfonate or from solutions containing known amounts of ⁇ -glucuronidase.
  • a kinetic determination of ⁇ -glucuronidase activity by absorption can also be made using 1,3,6-pyrene trisulfonate-8- ⁇ -D- glucuronide [VII].
  • a 50 ⁇ L sample which contains ⁇ -glucuronidase is added to 1.0 mL of a 0.1 mM solution of 1,3,6-pyrene trisulfonate-8- ⁇ -D-glucuronide [VII] in a 0.1 M solution of sodium acetate in deionized water (pH 4.25), mixing thoroughly, and measuring optical density at 410 nm continuously in a temperature controlled cell.
  • the initial linear rate of increase in absorbance is proportional to the enzyme activity of the sample.
  • a standard curve can be prepared from data collected from solutions containing known amounts of 8-hydroxy- 1,3,6-pyrene trisulfonate or from solutions containing known amounts of ⁇ -glucuronidase.
  • a fluorescence endpoint determination of ⁇ -galactosidase activity can be made using 1,3,6-pyrene trisulfonate-8- ⁇ -D- galactoside as a substrate.
  • 1,3,6-pyrene trisulfonate-8- ⁇ -D- galactoside is prepared as set forth in Example 4 below, and is of the formula [VIII]:
  • 1,3,6-PYRENE TRISULFONATE-8- ⁇ -GALACTOSIDE To make this determination, 50 ⁇ L samples which contain ⁇ - galactosidase are added to 0.1 mL of a 1.0 mM 1,3,6-pyrene trisulfonate-8- ⁇ -D-galactoside [VIII] solution in 0.1 M 4- morpholinepropanesulfonic acid in deionized water. After incubation for 15 minutes at ambient temperature of about 22°C, the reaction is quenched by adding 2.0 mL of 0.25 M glycine, pH 10.5, vortexing each solution thoroughly, and measuring fluorescence emission at 515 nm after excitement in a fluorimetric cuvette at 454 nm.
  • Emission at 515 nm is compared with a plot of intensity of emission at 515 nm as a function of 8-hydroxy-l,3,6-pyrene trisulfonate concentration or with a plot of intensity of emission at 515 nm as a function of ⁇ -galactosidase concentration.
  • a fluorescence endpoint determination of ⁇ -glucosidase activity can be made using 1,3,6-pyrene trisulfonate- 8 - ⁇ -D-glucoside as a substrate.
  • 1,3,6-pyrene trisulfonate-8- ⁇ -D-glucoside is prepared as set forth in Example 3 below, and is of the formula [DC]:
  • Emission at 515 nm is compared with a plot of intensity of emission at 515 nm as a function of 8-hydroxy-l,3,6-pyrene trisulfonate concentration or with a plot of intensity of emission at 515 nm as a function of ⁇ -glucosidase concentration.
  • Examples 1 and 3 demonstrate the production of pyrene-(l,3,6-trisulfonic acid)-8- ⁇ -D-glucuronide [VII] and of pyrene-(l ,3,6-trisulfonic acid)-8- ⁇ -D-glucoside [IX], respectively, compounds which differ from one another only with respect to the identity of the substituent on the 5 carbon, carboxyl in the former and CH2OH in the latter.
  • the glucuronide was produced from bromo- 2,3,4-tri-O-acetyl- ⁇ -D-glucopyranuric acid methyl ester (methyl (tri- 0-acetyl( ⁇ )-D-glucopyranosyl bromide)-uronate [IV]), which is commercially available, and can also be produced by a method disclosed in BoUenback et al., supra.
  • the glucoside was produced from 2,3,4,6-tetra-O-acetyl- ⁇ -D-glucopyranosyl bromide, which is also available commercially and, in addition, can be produced by the method of Koenigs and Knorr, Berichte der Deutschen Chemischenmaschine. Vol.
  • a saccharide starting material that has a lactone or a carboxy group can be reacted to produce a methyl or another ester which is then reacted with the acetyl bromide, as in BoUenback, et al., to produce the corresponding O-acetyl bromide ester, which is then reacted with the pyrene sulfonate in the presence of the tetradecyl-trimethyl ammonium bromide or an equivalent.
  • the tetradecyl-trimethyl ammonium bromide, or a suitable equivalent is used as a phase transfer catalyst in the synthesis.
  • the pyrene sulfonate reacts minimally with either the acetyl bromide or the acetyl bromide ester.
  • the O-acetyl bromide starting materials set forth in the Examples below can be produced from methyl ⁇ -D-glucuronate (Example 1), ⁇ -D- glucopyranose (Example 3), D-galactose (Example 4) and ⁇ -maltose (Example 5).
  • saccharides that can be reacted with the acetyl bromide to produce a polyacetylated bromocarbohydrate or that can be reacted to produce a methyl of another ester which is then reacted with the acetyl bromide, as in BoUenback, et al., to produce a polyacetylated bromocarbohydrate, include the other anomers of the compounds of the present invention, other naturally occurring and synthetic sugars, e.g., erythrose, threose, ribose, arabinose, xylose, lyxose, allose, altrose, mannose, gulose, idose, talose, sucrose and lactose, the last two, like maltose, being disaccharides, and cellobiose (a disacchari.de), xylan (a polysaccharide) and polyglucuronic acid- (a poly saccharide), the last three occurring in
  • the moiety represented by R in the formula of the compounds represented herein binds to one of the enzymes.
  • the determination of ⁇ -glucuronidase, ⁇ -galactosidase or ⁇ - glucosidase in a sample, or the 8-hydroxy-l,3,6-pyrene sulfonate equivalence of those samples, as described herein is the result of the enzyme binding to the glucuronide, to the galactoside or to the glucoside, respectively, which causes hydrolysis thereof and the release of free 8-hydroxy-l,3,6-pyrene trisulfonate.
  • the compounds of the present invention are also useful in other analytical test systems which require the measurement of the amount of enzyme present therein, particularly those analytical test systems employing enzyme-labeled assay reagents.
  • Such other analytical systems include, but are not intended to be limited to, heterogeneous and homogeneous enzyme immunoassays known in the art, such as competitive, sandwich and immunometric techniques where the amount of enzyme label in a particular fraction thereof can be measured.
  • immunoassay systems depend upon the ability of an immunoglobulin, i.e., a whole antibody or fragment thereof, to bind to a specific analyte wherein a conjugate comprising an antibody, or fragment thereof, to such analyte conjugated with a label or detectable moiety known in the art is employed to determine the extent of such binding.
  • detectable moieties or labels include, but are not intended to be limited to, enzymes, chromogens, luminescent compounds, fluorescent compounds, and the like.
  • the extent of binding is determined by the amount of the detectable moiety present in the conjugate which either has or has not participated in a binding reaction with the analyte, wherein the amount of the detectable moiety detected and measured can be correlated to the amount of analyte present in the test sample.
  • the ⁇ -galactosidase activity of the conjugate can be determined by adding a substrate solution comprising 2 mM 1,3,6- pyrene trisulfonate-8- ⁇ -D-galactosidase, 1 mM magnesium chloride, and 10 mM 2-mercaptoethanol in 0.1 M potassium phosphate buffer, pH 7.0. Subsequent to the addition of the substrate, the enzyme- catalyzed hydrolysis is stopped with the addition of a volume of 2 M glycine, pH 10.5.
  • a test kit according to the present invention comprises all of the essential reagents required to perform an assay for the determination of a glycohydrolytic enzyme as described herein.
  • the test kit is presented in a commercially packaged form as a combination of one or more containers holding the necessary reagents, as a composition or admixture where the compatibility of the reagents will allow.
  • test kit comprises a compound of the formula [VI], and may also include other materials as are known in the art and which may be desirable from a commercial user standpoint, such as buffers, diluents, standards, and the like.
  • a particularly preferred test kit is for the determination of ⁇ - glucoronidase for the diagnosis of periodontal disease wherein such test kit comprises the compound of the formula [VII].
  • Pyrene-(l,3,6-trisulfonic acid) -8 - ⁇ -D -Glucuronide [VII] was prepared from 8-hydroxy-l,3,6-pyrene trisulfonic acid trisodium salt (Eastman Kodak Co., Rochester, New York, USA) and bromo-2,3,4,-tri-0-acetyl- ⁇ -D-glucopyranuronic acid methyl ester (Sigma Chemical Co., St. Louis, Missouri, USA).
  • the blue fluorescent fractions contained an acetyl protected intermediate product of the compound of formula [VII] wherein the CO2H group was CO2CH3, the three hydroxyls were OOCCH3 groups, and the three hydrogens were sodiums) were pooled and dried by rotoevaporation.
  • the dried, acetyl protected intermediate product was dissolved in the sodium methoxide solution, and the solution was allowed to stand at room temperature of about 22°C for 4 hours, after which time an addition of 2.5 mL deionized water was made, and the solution was mixed thoroughly and allowed to stand at room temperature for about 15 hours.
  • Cold acetone was then added to the solution to precipitate crude 1,3,6-pyrene trisulfonate-8- ⁇ -D- glucuronide [VII], which was further purified by dissolving in a small amount of deionized water and precipitating with cold methanol; the final product was recovered by filtration and dried, yielding 0.4 g product, 17 percent of theory.
  • Solutions containing known amounts of 8-hydroxy-l ,3,6- pyrene trisulfonate were prepared, and 50 ⁇ L portions of each solution were mixed with 2.1 mL of 0.25 M glycine (pH 10.5) and placed in fluorimetric cuvettes and excited at 454 nm. Emission at 515 nm was measured for each solution, and the data were used to prepare a plot of intensity of fluorescence emission at 515 nm as a function of 8-hydroxy-l,3,6-pyrene trisulfonate concentration.
  • Solutions containing known amounts of ⁇ -glucuronidase were also prepared, and were used to determine fluorescence at 515 nm as a function of ⁇ -glucuronidase concentration; 50 ⁇ L portions of each solution were added to 0.1 mL of 0.3 mM 1,3,6-pyrene trisulfonate- 8- ⁇ -D-glucuronide [VII] (prepared according to Example 1) in a 0.1 M sodium acetate solution in deionized water, and, after incubation for 15 minutes at ambient temperature of about 22°C, the reactions were quenched by adding 2.0 mL of 0.25 M glycine, pH 10.5.
  • Samples containing ⁇ -glucuronidase were analyzed by adding 50 ⁇ L of each to 0-.1 mL of a 0.3 mM solution of 1 ,3.6-pyrene trisulfonate- 8- ⁇ -D-glucuronide [VII] in a 0.1 M sodium acetate solution in deionized water, and, after incubation for 15 minutes at ambient temperature of about 22°C, quenching the reaction by adding 2.0 mL of 0.25 M glycine, pH 10.5, vortexing each solution thoroughly, and measuring fluorescence at 515 nm after excitation in a fluorimetric cuvette at 454 nm.
  • the 8-hydroxy-l,3,6-pyrene trisulfonate equivalence was determined by reference to the first curve (Table 1), while the ⁇ - glucuronidase content was determined by reference to the second (Table II).
  • Example 3 The procedure described above in Example 3 was followed to produce 1 ,3,6-pyrene trisulfonate-8- ⁇ -D-galactoside [XIII] by substituting 0.6 grams 2,3,4,6-tetra-O-acetyl- ⁇ -D-galactosyl bromide for bromo-2,3,4,6-tetra-0-acetyl- ⁇ -D-glucopyranosyl bromide in the procedure of Example 3.
  • Example 3 The procedure described above in Example 3 was followed to produce 1,3,6-pyrene trisulfonate-8- ⁇ -D-maltoside [X] by substituting 1.2 g ⁇ -bromohepta-O-acetylmaltose for bromo-2,3,4,6- tetra-O-acetyl- ⁇ -D-glucopyranosyl bromide in the procedure of Example 3.

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Abstract

L'invention concerne de nouveaux dérivés de trisulfonate de pyrène et leur utilisation selon un procédé de détermination de l'activité d'une enzyme glycohydrolytique. Le procédé consiste à (a) préparer une solution à examiner comprenant un échantillon à examiner contenant l'enzyme glycohydrolytique et un dérivé de trisulfonate de pyrène selon la présente invention, ce dérivé étant hydrolisé par l'enzyme glyclohydrolitique pour former un trisulfonate libre de 8-hydroxy-1,3,6-pyrène qui est fonction de la quantité d'enzyme glycohydrolytique présente dans l'échantillon à analyser et qui peut être mise en corrélation avec ladite quantité; et (b) à mesurer soit l'intensité de la fluorescence, soit l'intensité optique de la solution à analyser et à mettre en corrélation la valeur obtenue avec la présence ou la quantité d'enzyme glycohydrolytique dans l'échantillon à analyser. Un dérivé préféré de trisulfonate de pyrène est un pyrène-(acide 1,3,6-trisulfonique)-8-β-D-glucuroconjugué utilisé pour la détermination de l'activité de la β-D-glucoronidase dans le diagnostic d'une paradontolyse.
PCT/US1993/000988 1992-01-29 1993-01-28 Reactif et procede pour la determination de l'activite d'enzymes glycohydrolitiques WO1993015097A1 (fr)

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US07/827,669 US5272260A (en) 1992-01-29 1992-01-29 Reagents and methods for the determination of glycohydrolytic enzymes
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000003034A3 (fr) * 1998-07-10 2000-04-27 Chromagen Inc Nouveaux substrats fluorogenes pour enzymes hydrolytiques
WO2007024945A1 (fr) 2005-08-25 2007-03-01 Novartis Ag Derives imidazolo condenses utilises pour inhiber l'aldosterone synthase et l'aromatase

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US6534637B2 (en) 2001-02-12 2003-03-18 Beckman Coulter, Inc. Synthesis of chlorophenol red glucuronic acid
US20060088722A1 (en) * 2004-10-25 2006-04-27 Aller Robert C Optical pH sensor
US7951603B2 (en) * 2005-03-31 2011-05-31 The Noguchi Institute Method of mass spectrometric analysis of saccharides with aldehyde groups using labels containing a pyrene ring and a hydrazide group

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Publication number Priority date Publication date Assignee Title
WO2000003034A3 (fr) * 1998-07-10 2000-04-27 Chromagen Inc Nouveaux substrats fluorogenes pour enzymes hydrolytiques
US6635435B1 (en) 1998-07-10 2003-10-21 Chromagen, Inc. Fluorogenic substrates and their use
US6949632B2 (en) 1998-07-10 2005-09-27 Chromagen, Inc. Fluorogenic substrates
WO2007024945A1 (fr) 2005-08-25 2007-03-01 Novartis Ag Derives imidazolo condenses utilises pour inhiber l'aldosterone synthase et l'aromatase

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